Antibiotics antimycotic solution

L-dopa and acetylcholinesterase activity assay kit (Cat# CS0003) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Folin–Ciocalteu phenol reagent and aluminum chloride were obtained from Loba Chemie (Mumbai, India). Quercetin (98% purity) was purchased from Chanjao Longevity Co., Ltd. (Bangkok, Thailand). P19 cell line ATCC CRL-1857 was obtained from American Type Culture Collection, USA. Alpha minimal essential medium (α-MEM), fetal bovine serum (FBS), newborn calf serum (NCS), and antibiotics-antimycotic solution were purchased from Gibco, USA. All trans-retinoic acid, cytosine-1-β-D-arabinoside, 1:250 porcine trypsin, poly-L-lysine (MW > 300,000), XTT (2,3-bis(2-methoxy-4-nitro-5-sulphonyl)-2H-tetrazolium-5-carboxanilide sodium), and phenazine methosulfate (PMS) were obtained from Sigma, USA. Dimethylsulfoxide (DMSO) and methanol analytical grade were purchased from Merck, Germany. A total of 96-well plates were purchased from Corning, USA. A 100-mm Bacteriological culture dish was obtained from Hycon, USA.

Cells were cultured in RPMI 1640 containing 10% (vol/vol) FBS, 50 µM 2-mercaptoethanol, 1% (vol/vol) non-essential amino acids, 10 mM HEPES, and 1% (vol/vol) antibiotics/antimycotic solution (all from Invitrogen). For TCR stimulation, the cells were incubated for the indicated times with plate-bound anti-CD3ε mAb (0.1, 1, or 10 µg/ml; PeproTech) only or in combination with plate-bound anti-CD28 mAb (1 or 10 µg/ml; PeproTech). For cytokine stimulation, IL-7 (20 ng/ml; R&D Systems), TGF-β (10 ng/ml; R&D Systems), IL-6 (20 ng/ml; R&D Systems), IL-1β (10 ng/ml; PeproTech), and IL-23 (20 ng/ml; PeproTech) were added to the medium.

The spleen was collected, minced and filtered through a 70-μm nylon cell strainer (Corning, New York, USA) to obtain a single cell suspension. The splenocytes were then suspended in 5 mL of RPMI 1640 containing heat-inactivated 5% (vol/vol) FBS and 1% (vol/vol) antibiotics/antimycotic solution (all from Invitrogen) and centrifuged at 300 × g for 3 min at 4 °C. Then, the pellet was treated with 1 mL of ACK lysing buffer (Thermo) incubated for 3 min at room temperature and centrifuged at 300 × g for 3 min at 4 °C. The pellet was washed and re-suspended in medium and filtered through a 70-μm strainer.

Dulbecco’s modified Eagle medium (DMEM) culture medium, trypsin, fetal bovine serum (FBS), and antibiotics/antimycotic solution were procured from Invitrogen, Life Technologies, Grand Island, NY, United States. Plastic wares and other consumables used in the study were purchased from Nunc, Roskilde, Denmark. H₂O₂ and all specified chemicals and reagents were purchased from Sigma Chemical Company Pvt., Ltd., St. Louis, MO, United States.

Nepeta deflersiana (Lamiaceae) plants were collected from Shaza Mountains, Saudi Arabia. The identity of the plant was confirmed by Dr. Jakob Thomas, KSU, and a voucher specimen (#15797) was deposited in the herbarium. Cell culture medium, antibiotics-antimycotic solution, trypsin, and FBS were procured from Invitrogen, USA. Plastic wares and other consumables were obtained from Nunc, Denmark. Other chemicals/reagents used in this study were purchased from Sigma, USA.

PCa cells LNCaP, 22Rv1, PC3 and DU145 and healthy prostate cells RWPE-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cell media RPMI-1640 and Keratinocyte-SFM (K-SFM) were acquired from Fisher Scientific (Pittsburgh, PA). Fetal Bovine Serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA). Antibiotics-Antimycotic solution (AA) was purchased from Thermo Fisher Scientific (Waltham, MA). LNCaP, 22Rv1, PC3 and DU145 cells were cultured in RPMI-1640 media supplemented with 10% FBS and 1% AA. RWPE-1 cells were cultured in K-SFM media supplemented with bovine pituitary extract (50 μg/mL), human epidermal growth factor (5 ng/mL) and 1% AA. All cell lines were maintained in a humidified atmosphere at 37 °C with 5% CO₂ air.

Human Rheumatoid arthritis FLSs (HFLSs-RA) and Normal FLSs from BeiNa Biological Technology Co., Ltd were cultured in DMEM containing 10% FBS and 1% antibiotics/antimycotic solution (all from Thermo Fisher Scientific) at 37°C, and 5% CO₂ in a humidified atmosphere incubator.